1. Technical Field
This invention relates generally to nucleic acid analysis and more specifically to the label-free DNA analysis by surface hybridization to morpholine monolayers.
2. Prior Art
Surface hybridization, in which sequence-specific binding between polynucleic acid “probes” on a solid support and complementary “targets” from solution occurs at a solid-liquid interface, was introduced as a diagnostic method in the 1960's1,2. The technique continues to be widely exploited in modern DNA microarray and biosensor technologies for genotyping, transcriptome profiling, genetic identification, and related diagnostic applications3. When hybridization occurs at a surface, experiments show that the phenomenology of the reaction is more complex than in solution4-17. The crowded interfacial environment is characterized by nucleotide concentrations that approach the molar range, and the resultant amplification of interactions between nucleotides can have a dramatic impact on physical behavior manifesting, for example, in suppressed binding affinities orders of magnitude lower than those in solution6,14,15,17,18.
A consequence of molecular crowding is that a DNA probe layer presents a high, ˜0.1 mol L−1 concentration of immobilized negative charge. This charge density erects an electrostatic barrier to entry of like-charged would-be hybridization partners from solution. In order for hybridization to proceed, this barrier needs to be screened through the addition of salt such that the solution number density of mobile ions becomes comparable to that of the surface-bound DNA charge17. While this electrostatic screening benefits hybridization, it also suppresses electrostatic interactions between the probe layer and the underlying support that could be used to control or to monitor the surface hybridization state. As an alternative approach that avoids this drawback, electrostatic hindrance to surface hybridization can be tempered through the use of neutral (i.e. uncharged) probes, such as peptide nucleic acids (PNAs)19,20 and Morpholinos21. Moreover, because the probe layer starts from an uncharged state, binding of charged nucleic acid targets is expected to elicit stronger structural changes, thus enhancing prospects for analysis of the hybridization reaction through purely electrostatic means.
From the selection of neutral probes, the high binding affinity of PNAs provides strong mismatch discrimination19,22 that is expected to be well suited to genotyping and to resequencing. Applications of PNAs have typically relied on 16 mer or shorter sequences23-26 since longer strands, or ones containing long stretches of pyrimidines and purines, become increasingly challenging to prepare27-29 and have greater potential for cross-reactivity with mismatched sequences. PNAs are thus expected to be less well suited to applications such as gene expression and pathogen detection which benefit from longer probe lengths, up to 70 nt,30,31 to provide robust identification of a target's unique origin (i.e. a specific gene or biological entity). In such instances Morpholinos, which place few constraints on sequence design or length, are expected to be advantageous. Morpholinos also mitigate some of the difficult physicochemical properties of neutral DNA analogues; for example, they are about 100-fold more soluble than comparable PNAs and their relatively stiff backbone reduces propensity toward self-aggregation32.